Session 7: Environmental Responses
Chair: X-W Deng, Yale University, USA.
email:pfd1@le.ac.uk
Summary: Paul Devlin, Leicester University, UK.
The environmental responses section brought together speakers from a range of disciplines. In particular, the session reflected mostly the current dynamism within the field of photomorphogenesis. One talk was on cold-tolerance.Xing-Wang Deng from Yale University, chairing the session presented the latest results from the work in his lab focusing on the interactions between the various elements involved in the regulation of light-responsive genes. Mutants deficient in one or more of the COP proteins display constitutive photomorphogenesis. A number of COP genes have been cloned which has allowed an impressive analysis of the interactions between the various COP proteins. Several of the COP proteins (COP8, COP9 and COP11) have been demonstrated to be part of a larger nuclear-localised complex, whilst another, COP1, has been demonstrated to interact with the HY5 protein, a putative trans-acting factor implicated in the phytochrome / blue-light receptor signal transduction pathways (see also Oyama's talk below). Interestingly, a functional GUS-COP1 fusion protein has shown that COP1 is localised in the nucleus darkness but excluded from the nucleus in light. Several of the cop mutations (cop8-15) affect this nuclear localisation of COP1 suggesting that this may hold the key to the cop phenotype.
A second group of proteins involved in the suppression of light-activated genes were discussed by M. Chatterjee from Joanne Chory's lab at the Salk Institute. The DET proteins (de-etiolated) add weight to the view that the transduction of the light signal involves a "complex network of interactive signalling components". Mutants deficient in DET1 also appear to posses aberrant circadian rhythms suggestive of a disruption of the input to the clock. The mutation ted1 suppresses det1 and analysis of ted1 may provide further insight into the role of DET1. Research into the function of the DET2 protein has already yielded results. DET2 encodes a steroid 5a-reductase brassinosteroid biosynthesis enzyme and it has been demonstrated that the det2 phenotype can be rescued by exogenous application of brassinolide.
Steven Streatfield, also from Joanne Chory's lab, presented analysis of one of a group of positive regulators of light inducible genes. Induction of CAB expression by red, blue or white light is reduced in cue1 mutants. The CAB under-expression phenotype of cue1 involves an interaction with the proximal region of the CAB promoter which is known to confer light regulation on the CAB gene. DNA-binding activity within the CAB promoter is currently being compared in wild-type and the cue1 mutant.
My own talk focused on my work in Gary Whitelam's lab at Leicester University on the perception of the light signal or, more specifically, the roles of the various phytochrome species involved. Analysis of mutants that are null for phytochome A and phytochrome B has revealed that these two phytochromes have discrete but overlapping roles in the regulation of photomorphogenesis. The phyA, phyB double null mutant still displays several phytochrome-mediated responses suggesting regulation by phytochrome C, D or E. Specifically I have demonstrated that phytochrome action is required for the rosette habit of Arabidopsis and that a novel phytochrome (C, D or E) controls internode elongation and flowering time. A mutant deficient in this novel phytochrome is predicted to be early flowering and to possess elongated internodes. Mutant screening in the phyA, phyB double background has yielded several mutants which have this phenotype and which also lack some additional phytochrome-mediated responses. Finally, the phenotype of a new mutant gn7, the result of a large deletion including the whole of the PHYC gene was described.
Gareth Jenkins from Glasgow University presented an analysis of the promotion of chalcone synthase expression (CHS) by blue and UV light. CHS induction involves distinct blue/UV-A and UV-B pathways. Studies with the hy4 mutant indicate that the CRY1 photoreceptor mediates the response to blue/UV-A but not UV-B. Blue and UV-A light were shown to act synergistically with UV-B to promote CHS expression. Experiments on CHS induction in cell cultures indicate that induction by blue, UV-A and UV-B involves protein phosphorylation and release of calcium from internal stores. However, the calmodulin antagonist W7 blocks UV-B induction but not blue induction. A screen for mutants with altered CHS expression was outlined. One such mutant, icx1, with increased CHS expression has been previously described. A similar mutant was introduced which demonstrates increased pigmentation and an increased tolerance to UV-B with respect to DNA damage.
Dave Somers from Steve Kay's lab at Virginia University gave the first of two talks on the genetic analysis of circadian rhythms in Arabidopsis. Andrew Millar, now at Warwick University, has carried out a screen for mutants with altered circadian regulation of a cab-luciferase transgene. Twenty one such mutants have been isolated. Dave's talk focused on one of these mutants toc1 which has a short period for cab-luciferase cycling, for the leaf movement rhythm and for the stomatal conductance rhythm. toc1 forms a strong candidate for a clock component. Cloning of TOC1 and transgenic alteration of its expression will allow this to be tested.
The perception of daylength, a central factor in the control of flowering time, is thought to be mediated by a circadian sensitivity to light. Isabelle Carre from Warwick University discussed an assay to determine whether the clock which controls overt circadian rhythms in Arabidopsis is also involved in the perception of daylength. Photoperiod sensitivity was examined in mutants with altered circadian rhythms. The response to a single inductive long day of different lengths was used as an indicator of photoperiod sensitivity. The toc1 mutant flowered early in all treatments but it can not be ruled out that it has a shorter critical daylength than that used in the experiment. The elf3 mutant is early flowering and photoperiod insensitive. elf3 is also arrhythmic for cab-luciferase expression. It is proposed that ELF3 forms a component of the input pathway to the clock. If so it is possible that the effect of elf3 on flowering is independent of the effects on the clock. Similarly, leaf movement rhythms were examined in mutants with altered sensitivity to photoperiod. esd4 is a short period mutant whilst in the fun mutants flowering is uncoupled from daylength. Both, however, display normal leaf movement rhythms.
Tokitaka Oyama working with Kiyotaka Okada at Kyoto University presented an analysis of HY5. The hy5 mutant, isolated by Maarten Koornneef displays a long hypocotyl in red, blue and far-red light leading to the proposal that HY5 acts downstream of multiple photoreceptors. However, it was demonstrated here that HY5 is involved not only in the control of hypocotyl length but also in the development and gravity responses of roots. A T-DNA-tagged hy5 mutant was isolated independently on the basis of the abnormal root development. This allowed the cloning of the HY5 gene which encodes a protein with basic - leucine zipper motifs suggesting that it functions as a trans-acting factor.
Another locus involved in hypocotyl elongation was introduced by Herman Höfte from INRA. The procuste1 (prc1) mutant has a short hypocotyl in darkness. However, in light prc1 displays normal hypocotyl elongation. Double mutant analysis has demonstrated that phytochrome but not cryptochrome is required for this reversal. It was concluded that two genetic pathways exist in the control of hypocotyl cell elongation. Phytochrome mediates a switch from a PRC1-dependent pathway acting in etiolated seedlings to a PRC1-independent pathway.
Paul Davison from John Allen's lab at Lund University discussed a method for measuring chlorophyll fluorescence, an indicator of photosynthetic efficiency. A screen for mutants with aberrant chlorophyll fluorescence in response to specific light sources was described. Such a phenotype would result from an inability to adapt to specific light conditions and would provide a tool to analyse this adaptation.
Finally, Gary Warren from Imperial College, London described several mutants unable to tolerate freezing following cold acclimation. For five of these sensitive to freezing (sfr) mutants, it was demonstrated that the phenotype is not due to the levels of sugars, fatty acyl composition or a general lack of cold-induced expression. Progress in the isolation of these SFR genes was presented.